Nerve growth factor (NGF) was first described by Levi-Montalcini (J. Exp. Zool. 116:321-362, 1951) as an activity secreted by a mouse sarcoma tumor implanted into a chick embryo. Both sensory ganglion and sympathetic ganglion neurons grew neurites into the sarcoma, which also supported the growth of peripheral neurons in culture. The factor, purified to homogeneity from mouse submandibular glands in 1956 by Levi-Montalcini and Cohen (Proc. Natl. Acad. Sci. USA 42:571, 1956) consists of a complex (referred to as 7S NGF, from its sedimentation coefficient) comprised of three different subunits. NGF's neurotrophic activity resides entirely within the .beta.-subunit (hereinafter referred to as NGF), a dimer consisting of two equivalent monomers of approximately 13,000 dalton molecular weight.
A role for NGF as a neurotrophic factor in the peripheral nervous system (PNS) was rapidly established through both in vitro and in vivo experiments (Levi-Montalcini and Angeletti, Physiol. Rev. 48:534-569, 1968; Johnson, et al., Science 210:916-918, 1980). These studies demonstrated that sympathetic neurons of the PNS have an absolute requirement for NGF for survival throughout life, while many sensory neurons require NGF during certain periods of development. As an extrapolation of these early findings, NGF and related neurotrophic factors have been shown recently to be useful in the treatment of sensory and autonomic neuropathies (Kaplan, et al., Science 252:554-558, 1991). More specifically, NGF prevents the development of small-fiber sensory neuropathies that result from the use of taxol, a chemotherapeutic agent (Apfel, et al., Ann. Neurol. 29:87-90, 1991). NGF is also efficacious against the development of large-fiber sensory neuropathies resulting from the anticancer drug cisplatin, and in an animal model of diabetes-induced neuropathy (Apfel, et al., Ann. Neurol. 31:76-80, 1992) .
NGF is synthesized in the periphery by the non-neuronal target tissues innervated by the NGF-dependent neurons. Upon binding of NGF to its receptor, the NGF receptor complex is internalized by the neuron and retrogradely transported back to the neuron cell body. NGF's intracellular mechanism of action is not yet fully understood.
It was not until 1983 that NGF was detected in the central nervous system (CNS) (Ayer-LeLievre, et al., Medical Biology 61:296-304, 1983). This discovery was preceded by the demonstration that the cholinergic neurons of the basal forebrain are responsive to NGF (Schwab, et al., Brain Res. 168:473-483, 1979). These neurons possess NGF receptors which are undistinguishable from the NGF receptors in the periphery. As in the PNS, NGF is synthesized by the target regions of the sensitive neurons, the hippocampus and the neocortex. NGF secreted by these target regions binds to its receptor on NGF-dependent neurons, and is required for the development and survival of these neurons. The NGF receptor, generally known as p75 because it has a molecular weight of about 75,000 daltons, has recently been cloned (Johnson, et am., Cell. 47:544-554, 1986).
In animal models, lesions of the septo-hippocampal neuronal pathway, which connects the neurons of the basal forebrain to the hippocampus, results in the degeneration of the neurons in the medial septal nucleus, due to the lack of trophic support that is normally provided by hippocampal NGF. This neuronal degeneration can be prevented by administration of NGF into the cerebrospinal fluid (CSF) of the animals via a miniosmotic pump [Gage, et al., J. Comp. Neurol. 269:147-155, 1988; Hagg, et al., Exp. Neurol. 101:303-312, 1988; Kromer, et al., Science 235:214-216, 1987]. NGF also enhances the ability of brain cholinergic neurons to survive a chemical insult. In animals with atrophied or lesioned basal forebrain cholinergic neurons, NGF has been shown to reverse the associated behavioral impairment (Fisher, et al., J. Neurosci, 11:1889-1906, 1991; Tuszynski, et al., Ann. Neurol. 30:625-636, 1991).
Even though there is no direct evidence for a reduced level of NGF in neurodegenerative disorders such as Alzheimer's disease (AD), the sensitivity of brain cholinergic neurons to NGF treatment is specially interesting, since these neurons are consistently depleted in AD. In fact, recent clinical findings in humans support the interpretation that treatment with NGF is useful in the treatment of AD (Olson, et al., J. Neural Transm. (D-P Sect) 4:79, 1992) .
NGF is a member of a family of related neurotrophic peptides, the neurotrophins, all with a high degree of similarity in terms of their molecular weight and amino acid sequence. Other representative members of the neurotrophins include brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). While these peptides are selectively expressed in discrete areas of the brain of mammals, they all seem to bind to the same p75 receptor as NGF (Squinto, et al., Cell 65:885-893, 1991), and they seem to possess neurotrophic activity at well-defined populations of neurons. For instance, BDNF exerts neurotrophic effects on substantia nigra dopaminergic neurons (Knuesel, et al., Proc. Acad. Sci. USA, 1991, (88), 961-965). Since degeneration of these neurons is directly responsible for Parkinson's disease (PD), it is conceivable that BDNF will be useful in the treatment of this devastating disease.
AD, PD and drug-induced peripheral neuropathies are but a few of the many neurodegenerative conditions that might be amenable to neurotrophic treatment. However, as mentioned above, NGF and related neurotrophins are large peptides (around 120 amino acids), which large size makes them unlikely therapeutic candidates. In addition to the lack of a ready source of endogenous peptide(s), these compounds possess very poor pharmacokinetic parameters (e.g. poor oral absorption, short in-vivo half-life) and administration to the target organs also represents a major problem. One solution requires the use of cerebral implants of genetically engineered cells (Rosenberg, et al., Science 242:1575-1578, 1988) or intracerebral pumping devices for continuous infusion of the peptide (Powell, et al., Brain Res. 515:309-311, 1990). For these reasons, it would be extremely useful to identify novel compounds with physicochemical properties different from the neurotrophins but still able to interact with the neurotrophins' p75 receptor. The ideal compounds would be non-peptides, since this would enhance in vivo half-life. The non-peptides would be much smaller in molecular size than the endogenous peptides, and this would be expected to enhance oral absorption and CNS penetration. The present invention describes the identification of such a group of compounds that bind to the p75 receptor, which binding may stimulate neuronal regeneration and thus be useful in the neurotrophic treatment of a variety of neurodegenerative disorders.